Recent research in human stem cells has shown a highly promising potential in transplantation therapy. Nevertheless, the sources for cloning human stem cells are limited and very difficult to control their purity and quality. In 1998, James Thomson et al. (e.g. U.S. Pat. No. 5,843,780, U.S. Pat. No. 6,200,806, U.S. Pat. No. 7,029,913, and No. 7,220,584) have isolated the first human embryonic stem (hES) cell line from the blastocysts of human embryos (Thomson et al., (1998) Science 282: 1145-1147). H1 and H9 are two typical cell lines derived from these isolated hES cells. Two years later, Gearhart et al. (e.g. U.S. Pat. No. 6,090,622, U.S. Pat. No. 6,245,566, and U.S. Pat. No. 6,331,406) also develop a means and method to isolate hES-like primordial germ cells from post-blastocyst human embryos. Because the way of these embryonic stem cell isolation methods must destroy the original embryos, many ethical and humanity concerns have been raised to debate the righteousness of using the hES cell lines so obtained for clinical therapy.
In recent years, more and more concerns about the safety issue of using the isolated hES cell lines have been noticed. For example, since the culture conditions for maintaining the long-lasting pluripotent stem cell state of these hES cell lines require some unidentified factors released from surrounding “feeder” fibroblast cells, the hES cells are usually cultured on a mouse or human fibroblast feeder layer. Prior arts attempting this approach include U.S. Pat. No. 6,875,607 to Reubinoff et al. However, the fibroblast feeder cells carry totally different antigen characteristics, which may very likely contaminate the hES cells and cause immune rejection in patients. Meantime, although some feeder-free culture conditions have also been developed, none of these feeder-free methods are capable of maintaining a stable, undifferentiated stem cell state of the hES cells for a long time. This problem is actually related to another drawback of the isolated hES cells, which is their impurity. None of the currently available hES cell lines can reach a 100% pure population in culture conditions. Even under the best feeder culture condition, an uncertain rate (about 3-5% or more) of the hES cells tend to differentiate into other cell types and lose their stem cell properties. One of the most frequently observed cell types differentiated from the hES cells is teratoma. Teratoma is a tumor derived from human germ line cells, often containing multiple cancerous-look cell types similar to embryonic endoderm, mesoderm and ectoderm tissues. Therefore, how to prevent the feeder contamination and increase stem cell purity are two main tasks for the present stem cell research.
Induced pluripotent stem (iPS) cells are newly introduced by Takahashi and Yamanaka in 2006 (Cell 126: 663-676). Using transgenically delivery of four transcription factor genes (Oct3/4, Sox2, c-Myc, Klf4) into mouse fibroblasts, they successfully reprogram and transform the somatic fibroblast cells into embryonic stem (ES)-like pluripotent cells in vitro. In 2007, the behavioral properties of these iPS cells are confirmed to be similar to those of the mouse embryonic stem (mES) cells (Okita et al., (2007) Nature 448: 313-317; Wernig et al., (2007) Nature 448: 318-324). Meantime, Yu et al. develop more new iPS cell lines from human fibroblast cells, using a similar approach with four other transgenes such as Oct4, Sox2, Nanog, and LIN28 (Yu et al., (2007) Science 318: 1917-1920). The utilization of iPS cells not only solves the ethical and impurity problems of the previous hES cells but also provides a potential patient-friendly therapy if in conjunction with the somatic cell nuclear transfer (SCNT) technology (Meissner et al., (2006) Nature 439: 212-215). Such an iPS cell-based SCNT therapy has been proven to be successful in treating sickle cell anemia in a transgenic mouse model (Hanna et al., (2007) Science 318: 1920-1923). Yet, the advance of iPS cell applications is not perfect. Two problems emerge during the processes of iPS cell generation; one is the use of retroviral transgenes and the other is the use of oncogenes (e.g. c-Myc). Retroviral transfection is the only effective means to simultaneously and transgenically deliver the four full-length genes into a targeted somatic cell, whereas the random insertion of retroviral vectors into the transfected cell genome may also affect other non-targeted genes and cause unexpected results. This is particularly dangerous when one or more of the delivered genes is an oncogene.
Simultaneous delivery of four full-length transgenes into one single cell is very difficult to control precisely. However, the iPS cell technology actually requires the use of multiple transcription factors to offset or coordinate the signal transduction among each other and certain other developmental factors. Although detail mechanism is still unclear, the combined gene effects of Oct4-Sox2-c-Myc-Klf4 or Oct4-Sox2-Nanog-LIN28 seemingly result in a cancellation of developmental signals required for early cell differentiation. Despite the embryonic stem marker Oct4, all other genes used in iPS cell generation are involved in certain developmental lineages. They are usually presented in different embryonic stages and/or locations to guide the specific cell differentiation. By misplacing them together, the disturbance of these developmental signals somehow stops the cell differentiation and then retreats the host cell back to an ES-like state until another new developmental signal is given again. This method works but is not natural. In natural fertilized eggs, maternal materials are responsible for the regulation of stem cell maintenance and replication. That is why embryonic cells before the 128-cell stage are all the same and all totipotent. Maternal materials are generated during oogenesis and deposited in a mature oocyte required for early embryonic development. In a mouse oocyte, RNAs occupy a large volume of maternal materials, corresponding to about 45% of the whole genomic transcriptome (Stitzel et al., (2007) Science 316: 407-408). During maternal-zygotic transition, these maternal RNAs are quickly degraded and the transcription of zygotic genes starts as early as at the two-cell stage to produce signals for further embryonic development (O'Farrell et al., (2004) Curr. Biol. 14: R35-45). It is conceivable that many of the maternal RNAs are inhibitors of the zygotic gene products in order to prevent developmental signals and maintain the totipotent/pluripotent cell division at the most early embryonic stage. Therefore, the secret of stem cell maintenance and renewal should reside in maternal materials rather than the developmental signals, which are shown much later than the pluripotent embryonic stem cell stage.
In sum, in order to generate and maintain human embryonic stem (hES)-like cells mimicking the natural way of maternal materials, a new strategy is highly desired for transgenically delivering the isolated maternal material(s) into a human stem or somatic cell, so as to maintain the stem cell property or to reprogram the somatic cell into a hES-like cell state. Therefore, there remains a need for an effective, simple and safe transgenic method as well as agent composition for generating hES-like cells, using maternal materials, particularly the maternal RNAs.